TRPA1-mediated repellency behavior in the red flour beetle Tribolium castaneum

The sensory perception of irritant chemicals results in escape and repellency behavioral patterns in insects. Transient receptor potential channels are cation channels that function as sensor proteins for several types of signals, such as light, sound, temperature, taste, as well as chemical and physical stimuli; among these, the TRPA channel is widely conserved and activated by irritant chemicals. Certain plant-derived essential oils (EOs), produced by secondary metabolism, are mixtures of volatile compounds, which are used as repellents because they contain environmentally sustainable ingredients. Citronellal, which is present in citronella EO from Cymbopogon species, is a potentially viable insect repellent; however, the repellency capability against coleopteran beetles remains limited. We investigated the citronellal-derived repellency behavior for the red flour beetle Tribolium castaneum, in which TcTRPA1 and odorant receptor co-receptor (Orco) expressions were mediated by RNA interference. Area-preference tests showed dose-dependent repellency behavior for citronellal; additionally, both TcTRPA1 and TcOrco double-strand RNA (dsRNA) micro-injection showed clear TcTRPA1 and TcOrco transcript reductions, and only TcTRPA1 dsRNA treatment significantly impaired repellency behavior. The relative expression level of the TcTRPA1 transcripts, evaluated by quantitative reverse-transcription PCR (qRT-PCR), revealed dominant expression in the antennae, indicating the antennae-expressed TcTRPA1-mediated repellency behavior.

Prior studies have been conducted on the molecular recognition mechanism of citronellal activity, in which insect TRPA1 is necessary to repel citronellal in two dipteran species, D. melanogaster and Anopheles gambiae mosquito. It is noteworthy that citronellal activated both TRPA1s when they were heterologously expressed in Xenopus oocytes 20,21 .
Alternatively, odorant perception derived from host plants is executed by peripheral odorant receptors (ORs) that are expressed on the dendrites of olfactory sensory neurons (OSNs) 22,23 . Insect ORs form heteromeric cation channels with the obligate odorant receptor co-receptor (Orco), which functions not only as a measure of odorant sensitivity but also as a chaperone for the localization and maintenance of ORs 24 . Therefore, Orco knockdown and/or null mutations result in critical olfactory loss of function 25 .
In Drosophila, two pathways are involved in the citronellal response 20 . One pathway is mediated by OR reception, because Orco mutation resulted in impaired repellent activity; the other pathway is the G-protein-coupled phospholipase C signaling cascade, in which the TRPA1 channel functions downstream, and the TRPA1 isoform, namely TRPA1(A), elicited citronellal-enhanced gustatory aversion 21,26 . Additionally, the TRPA1 isoform, namely TRPA1s, in mosquito species such as A. gambiae and Culex pipiens, is directly activated by citronellal 20,27 . It was described several TRPA1 isoforms in other insect species, to date, there are no reports about TRPA1 isoforms in T. castaneum.
In related to TRPA1 mediated chemoreception, it was reported that menthol, extracted from Mentha species, evokes nocifensive rolling behavior in Drosophila larvae, and that TRPA1 and TRPM are genetically interact and required for the manifestation of aversive behavior 28 . In vitro expression analysis showed that Drosophila TRPA1 was not activated by menthol 6 , underscoring the continuing debate on whether TRPA1 functions in the perception of menthol.
However, the repellency mechanism of citronellal against coleopteran beetles is yet to be elucidated. In addition, it remains unclear whether menthol stimulates the TRPA1-mediated repellency behavior of T. castaneum, although we previously reported that l-menthol evokes TcTRPM-mediated contact repellency behavior in T. castaneum 29 . The objective of the present study was to examine the repellency behavior of T. castaneum to citronellal using RNAi-mediated knockdown of TcTRPA1; this was aimed at determining whether TcTRPA1 participates in mediating the repellency response. We further examined the OR-mediated repellency behavior for citronellal and clarified the engagement of TcTRPA1 for menthol-derived repellency behavior in combination with TcTRPM.

Results
Repellency behavior for citronellal. The area-preference test revealed a dose-dependent repellency activity of citronellal against T. castaneum (Fig. 1). Citronellal showed high repellency activity at more than 2.5 μmol/cm 2 . Based on these results, we decided to set the concentration at 2.5 μmol/cm 2 for further analysis.  www.nature.com/scientificreports/ To clarify citronellal target, we cloned TcTRPA1 gene, and the full-length amino acid sequence showed high sequence identities (74-83%) with some coleopteran TRPA1s. The phylogenetic tree revealed a cluster in the coleopteran TRPA1s among insect TRPA1 channels (Supplementally Fig. S1).
Target transcripts suppression by RNAi was evaluated using quantitative reverse-transcription PCR (qRT-PCR). The TcTRPA1 double-stranded RNA (dsRNA) treatment successfully suppressed target TcTRPA1 transcripts (F 3,8 = 57.26, P < 0.001). The qRT-PCR analysis showed a reduction in the target TcTRPA1 transcripts to 27% (Fig. 2a). TcTRPA1 transcripts of solvent (H 2 O) and enhanced green fluorescent protein (EGFP) dsRNA treatments were not significantly different from those of non-injected beetles.
Behavioral studies of beetles treated with TcTRPA1 dsRNA revealed significantly altered repellency behavior. TcTRPA1 dsRNA injection significantly hampered the repellency behavior for citronellal (F 3,16 = 14.26, P < 0.001) (Fig. 2b). The repellency activities of the beetles injected with solvent or EGFP dsRNA were not significantly different from those of non-injected beetles.
The TcTRPA1 dsRNA-injected beetles were compared for standard synthetic repellent, N,N-diethyl-3-methylbenzamide (DEET) using the area-preference test. The beetles treated with the TcTRPA1 dsRNA showed repellency behavior for DEET, which was not significantly different from that of non-injected beetles (t-test, P = 0.084) ( Supplementary Fig. S2). qRT-PCR. TcTRPA1 was predominantly expressed in the antennae (F 4,10 = 337.00, P < 0.001) (Fig. 3a), and the transcript level was almost 34 times higher than that in the abdomen.

Relative expression level of TcTRPA1
To elucidate antennae-based citronellal detection by TcTRPA1, an area-preference test was performed on the de-antennated beetles. The results showed that the de-antennated beetles had a significantly reduced repellent index compared with the repellent index in the untreated beetles (t-test, P < 0.001) (Fig. 3b).
RNAi-mediated knockdown of TcOrco. The TcOrco dsRNA treatment was performed for further analysis of antenna-based citronellal detection specificity. The treated beetles showed a reduction in the target TcOrco transcripts to 25% (F 3,8 = 53.94, P < 0.001) based on the qRT-PCR results (Fig. 4a); the subsequent area-preference test showed that the repellency activity of citronellal was not significantly different from that of untreated beetles (t-test, P = 0.608) (Fig. 4b).
The relative expression profile of the TcOrco transcripts among different tissues confirmed that TcOrco was predominantly expressed in the antennae (F 4,10 = 350.60, P < 0.001) ( Supplementary Fig. S3).
Intervention of TcTRPA1 for l-menthol repellency. TcTRPA1 intervention for l-menthol repellency was clarified using RNAi. The TcTRPA1 dsRNA-treated beetles were not significantly different from the  Relative expression levels of TcTRPA1 transcripts in each abdomen, thorax, head, leg, and antenna specimen of adult Tribolium castaneum were revealed as fold change relative to the corresponding transcript levels seen in the abdomen (n = 3) analyzed by qRT-PCR. Data are expressed as mean ± standard error of the mean (SEM). The same letters above the bars indicate no significant difference at P > 0.05 (one-way ANOVA and Tukey-Kramer HSD tests). (b) Mean repellent index of untreated and de-antennated adult beetles treated with 2.5 μmol /cm 2 of citronellal (n = 5). Data are expressed as mean ± SEM, and *** indicates that there was significant difference between them (*** P < 0.001, two-tailed student's t-test). TRP: transient receptor potential.  Fig. S4).

Discussion
The TRPA1 channel is widely expressed in insects, and TRPA1 is activated by noxious heat and irritant chemicals. To elucidate the molecular mechanism of citronellal aversion in T. castaneum, RNAi-based knockdown of TcTRPA1 was employed and repellency movement was evaluated. The results clearly showed that the TcTRPA1 dsRNA-treated beetles were not repelled by citronellal in the area-preference test, demonstrating the TcTRPA1mediated repellency behavior for citronellal. The synthetic repellent compound DEET, N,N-diethyl-3-methylbenzamide, remains the gold standard. DEETbased repellency involves multiple pathways and mechanisms; however, it remains an area of contention 30 . In our prior studies, T. castaneum showed repellency behavior for DEET 29,31 ; however, to the best of our knowledge, . TcOrco did not mediate the citronellal-based repellency behavior of Tribolium castaneum. (a) TcOrco dsRNA was micro-injected into the pupae; after the adult emerged, the targeted transcripts were measured by qRT-PCR. The expression level was revealed as fold change relative to the expression level seen in the noninjection beetles (n = 3). Data are expressed as mean ± standard error of the mean (SEM). The same letters above the bars indicate no significant difference at P > 0.05 (one-way ANOVA and Tukey-Kramer HSD tests). (b) The mean repellent indices for citronellal were compared in the area-preference test (n = 5). Data are expressed as mean ± SEM, and N.S. indicates there was no significant difference between non-injected and TcOrco dsRNA treated beetles (P = 0.608, two-tailed student's t-test). Orco: odorant receptor co-receptor. there are no studies that report that DEET activates insect TRP channels. We analyzed the area-preference test with TcTRPA1 dsRNA treatments for DEET repellency, and the repellent index of the TcTRPA1 dsRNA-treated beetles did not show a significant difference from that of the untreated beetles, implying the specific interaction of citronellal against TcTRPA1. The adult TcTRPA1 transcript expression profile in the various body parts revealed that the expression in the antennae was dominant; a similar case was observed in the hemipteran order, in the brown planthopper Nilaparvata lugens, which showed high NlTRPA1 expression in the antennae 32 . From these results, we proposed that T. casteneum repels citronellal with TcTRPA1-mediated antenna perception. We removed the antennae and compared the repellent index with that of the untreated beetles and showed a significant reduction in the repellent index by de-antennated treatment. Meanwhile, in our previous study, contact repellency activity of l-menthol did not show a significant difference between de-antennated and untreated beetles 29 , these results indicated antenna-expressed-TcTRPA1-mediated repellency behavior for citronellal.
In the case of dipteran species, two pathways: one is indirect contribution of TRPA1 in G protein/phospholicase C signaling cascade, and the other is direct Orco contribution, for citronellal avoidance was revealed in D. melanogaster 20,21 . In the oriental fruit fly Bactrocera dorsalis, it was demonstrated that BdOrco dsRNA treatment resulted in the reduction of oviposition repellents induced by citronellal 33 . In mosquitoes, citronellal binds directly to TRPA1 in A. gambiae 20 . In lepidopterans, the TRPA1 channel of Helicoverpa armigera revealed that in vitro heterologous expression of Xenopus oocyte functions not only as a thermal sensor but also as an irritant chemical sensor, including citronellal 34 .
In contrast, in the case of Hemiptera, the western tarnished plant bug Lygus hesperus showed that although LhTRPA1 dsRNA injection reduced citronellal-induced avoidance movement, LhOrco dsRNA injection did not alter the response, suggesting an OR-independent pathway for citronellal repellency 35 . To clarify the OR-mediated citronellal repellency of coleopteran beetles, we next verified the TcOrco-mediated repellency for citronellal. The area-preference test revealed that TcOrco dsRNA-injected beetles did not show significant differences in repellency behavior from non-injected beetles. Since Orco is widely co-expressed with ORs and strongly conserved across insects 36 , the results suggested non-involvement of the OR perception system for citronellal perception in T. casteneum. In combination with these results, we propose that evolutionary diversity has arisen for citronellal perception in Insecta.
Multiple TRP channels have been suggested to mediate the perception of menthol 11,12 . In Drosophila larvae, menthol evoked TRPM-and TRPA1-dependent nocifensive rolling behavior, which is required for multidendritic class IV nociceptor neurons 28 . In that analysis, homozygous mutants, each of TRPM and TRPA1, showed significantly fewer rolled larvae in response to menthol; furthermore, heterozygous mutants of both TRPM and TRPA1 showed significantly inhibited rolling behavior for menthol, indicating a genetic interaction in menthol sensing. In contrast, the l-menthol repellency behavior of adult T. castaneum was affected by TcTRPM. The relative expression levels of TcTRPM showed minimal differences among various adult tissues such as abdomen, thorax, head, leg, and antenna 29 . However, the TcTRPA1 transcripts in the present study were predominantly expressed in the antennae, while the de-antennated adults still exhibited repellency behavior for l-menthol, suggesting that only TcTRPM was required for menthol-evoked repellency. In the case of mammals, except for the TRP channels, it has been revealed that menthol influences different kinds of voltage-gated ion channels and ligand-gated ion www.nature.com/scientificreports/ channels, such as GABA, glycine receptors, and nicotinic acetylcholine receptors 37 , suggesting the presence of different targets for the menthol-mediated repellency behavior in adult T. castaneum. We describe the antennae-expressed TcTRPA1-mediated repellency behavior by which T. castaneum responds to citronellal, our results suggest that coleopteran TRPA1 represents a potential target for sustainable insect repellents. Intriguingly, Tian et al. recently reported that D. melanogaster TRPγ, contained in TRPC subfamily, was directly activated by citronellal to initiate repellent behavior 38 , for which further experiments and screening are desirable even in coleopteran beetles.

Methods
Insects. Laboratory colonies of Tribolium castaneum were used in the present study. They were fed on wholegrain wheat containing 5% dried yeast and reared at 28 °C under dark conditions in an incubator.
Behavioral bioassay. Repellency behavior was evaluated using the area-preference method. Racemic citronellal was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) while l-menthol and N,N-diethyl-3-methylbenzamide (DEET) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The test compounds were dissolved in acetone at concentrations of 10, 25, and 50 mg/mL for citronellal; 25 mg/mL for l-menthol; and 10 mg/mL for DEET. Filter papers 90 mm in diameter (ADVANTEC No.2, Toyo Roshi Kaisha Ltd., Tokyo, Japan) were cut in half, and 500 μL of the solution was uniformly applied to half of the filter paper using a micropipette. The other half was treated with 500 μL of acetone as a control. Both filter papers were air-dried to completely evaporate the solvent and carefully rebounded using tape. Each reassembled filter paper was placed in a 90 mm glass petri dish. Twenty adult beetles were released into the center of each filter paper disk, and the glass petri dish was then covered and maintained at 28 °C under dark conditions. Each test was replicated five times. After 3 h, the beetle positions in each area were counted. The repellency activity of the compound was expressed in terms of the repellent index (RI), calculated according to the following formula: where N c is the number of beetles present in the control (solvent alone) area and N t is the number of beetles present in the treated area. cDNA cloning. The three adult T. castaneum were crushed together using a Biomasher II (Nippi Inc., Tokyo, Japan). Total RNA was extracted using the ReliaPrep RNA Cell Miniprep System (Promega Corporation, Madison, WI, USA), and first-strand complementary DNA (cDNA) was generated using ReverTra Ace α (TOYOBO Co., Ltd., Osaka, Japan). Full-length TcTRPA1 and TcOrco coding sequences were amplified by PCR using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Shiga, Japan) for TcTRPA1 and KOD plus Neo (TOYOBO) for TcOrco with the specific primers designed from the sequence data obtained from iBeetle-Base 39 (Supplementary Table S1). PCR was performed on a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: 35 cycles at 98 °C for 10 s, 58 °C for 15 s, and 68 °C for 4 min for TcTRPA1; 94 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 58 °C for 30 s, and 68 °C for 2 min for TcOrco. The PCR products were electrophoresed at 0.8% for TcTRPA1 and 1% for TcOrco agarose-gel, then purified using a MiniElute Gel Extraction Kit (QIAGEN, Hilden, Germany). The PCR product was assembled with the pUC19 plasmid vector using the In-Fusion HD cloning kit (Takara Bio) and transformed into competent DH5α Escherichia coli cells (ECOS Competent E. coli DH5α, NIPPON GENE, Tokyo, Japan). Plasmid DNA was purified using a Fast-Gene Plasmid Mini kit (NIPPON Genetics, Tokyo, Japan) and sequenced. BlastP search was performed using TcTRPA1 amino acids sequence, and maximum-likelihood (ML) phylogenetic analysis for the aligned sequence and the ML phylogenetic tree was constructed using MEGA X 40 . Bootstrap support was obtained using 1,000 bootstrap replications.
RNAi-mediated gene knockdown. The TcTRPA1 and TcOrco gene templates containing the T7 promoter sequence at the 5′ end were amplified with primers (Supplementary Table S1) using KOD Plus Neo (TOY-OBO) under the following conditions: 94 °C for 2 min, 40 cycles at 98 °C for 10 s, 58 °C for 30 s, and 68 °C for 1 min. The amplified templates were used for the synthesis of dsRNA using the RiboMAX Large-Scale RNA Production System T7 (Promega). The dsRNA products were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). EGFP dsRNA was synthesized from the pEGFP-C1 plasmid (Takara Bio USA, Inc., Mountain View, CA, USA) for the control, and TcTRPM dsRNA was prepared in the same manner as previously reported 29 . All dsRNA concentrations were adjusted to 2000 ng/μL using RNasefree water and injected into the pupal stage using a glass needle mounted with a micromanipulator (Narishige, Tokyo, Japan). After injection, the pupae were incubated in a 24-well plate set on a filter paper disk (ADVAN-TEC No.2) with a piece of whole-grain wheat with 5% of dried yeast at the bottom and incubated at 28 °C. After emergence, approximately 7-day-old adults were used for the area-preference test and subsequent qRT-PCR.
Quantitative analysis of transcription. To clarify RNAi-knockdown efficiency, total RNA was extracted from three adults after the area-preference test, and to compare relative expression level, total RNAs of each tissue (300 antennae, 30 heads, 20 thoraxes, 40 legs and four abdomens) from approximately 7-day-old adults were extracted in a similar manner, and cDNA was generated using ReverTra Ace qPCR RT kit (TOYOBO). Genespecific primers for qRT-PCR were designed using Primer3 (Supplementary Table S1). qRT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and THUNDERBIRD Next SYBR qPCR Mix (TOYOBO). The PCR amplification program consisted of 95 °C for 30 s, followed by 45 for melting curve analysis. The expression results were analyzed using the ∆∆Ct method. TcRPS6 gene (encoding ribosomal protein S6) was used to normalize gene expression, and mean Ct values for each gene were obtained from three replicates.

Statistical analysis
In the area-preference tests, the mean number of beetles in the sample area was compared with the number of beetles in the control area using a two-tailed paired t-test. The relative expression levels of the transcripts for dsRNA-treated beetles and each tissue, and the RI values were compared by one-way ANOVA, followed by the Tukey-Kramer HSD test. The RI values of antennae-dissected beetles, TcOrco dsRNA-treated beetles against citronellal, and TcTRPA1 dsRNA-treated beetles against DEET were compared using a two-tailed student's t-test. All statistical analyses were performed using R software.

Data availability
All data generated or analyzed during this study are included in this article and its supplementary information files.